Detecting single nucleotide polymorphism using overlapping hydrolysis probes

ABSTRACT

Methods for the rapid detection of the presence or absence of a SNP in a target nucleic acid in a sample are described. The methods can include performing an amplifying step, a hybridizing step utilizing a double stranded probe with two overlapping SNP specific hydrolysis probe sequences where one of the probe sequences can include a hairpin structure toward the 3′ end, and a detecting step. Furthermore, the double stranded SNP specific hydrolysis probes along with kits are provided that are designed for the detection of a SNP in a target nucleic acid.

FIELD OF THE INVENTION

The present invention relates to the field of polymerase chain reaction(PCR) based diagnostic, and more particularly, to PCR detection methodsutilizing overlapping hydrolysis probes.

BACKGROUND OF THE INVENTION

PCR is an efficient and cost effective way to copy or ‘amplify’ smallsegments of DNA or RNA. Using PCR, millions of copies of a section ofDNA are made in just a few hours, yielding enough DNA required foranalysis. This method allows clinicians to diagnose and monitor diseasesusing a minimal amount of sample, such as blood or tissue. Real-time PCRallows for amplification and detection to occur at the same time. Onemethod of detection is done by utilizing oligonucleotide hydrolysisprobes (also known as TaqMan® probes) having a fluorophore covalentlyattached, e.g., to the 5′ end of the oligonucleotide probe and aquencher attached, e.g., internally or at the 3′ end. Hydrolysis probesare dual-labeled oligonucleotide probes that rely on the 5′ to 3′nuclease activity of Taq polymerase to cleave the hydrolysis probeduring hybridization to the complementary target sequence, and result influorescent based detection.

Real time PCR methods can be used for amplifying and detecting sequencevariations in target nucleic acids having single nucleotide polymorphism(SNP). However, many of the available SNP detection/genotyping assaysare based on the assumption that the SNP is biallelic (see, e.g., Moritaet al., Mol. Cel. Probes, 2007, 21, 171-176). Detection of SNP withcurrently existing real time PCR methods lacks sufficient sensitivityand specificity. Hydrolysis probes, such as standard TaqMan® probes, aretypically designed to be about 18 to 22 bases in length in order to have8-10° C. higher melting temperature (Tm) as compared to the primer.Standard TaqMan® probes generally prove to be less specific andsensitive for SNP detection and fail to show complete discriminationbetween the WT (Wild-type) and the MT (Mutant) targets. Current TaqMan®based SNP genotyping assays involve the use of TaqMan® MGB (Minor GrooveBinders) probes that are shorter in length with increased probe-templatebinding stability for allelic discrimination. Additional basemodifications such as stabilizing bases (propynyl dU, propynyl dC) canalso be included in standard TaqMan® probe design for improved SNPdetection and discrimination. Thus there is a need in the art for aquick and reliable method to specifically detect SNPs in a sensitivemanner.

SUMMARY OF THE INVENTION

The subject matter of the present disclosure includes double strandedSNP specific hydrolysis probes where both probe strands are SNP specificand overlap at the SNP location. In some embodiments, one of the probestrands is designed to include a hairpin structure toward the 3′ end.The hairpin structure near the 3′end of the probe delays thehybridization of the 3′ portion of the probe to the template and thushelps in the discrimination of the WT and the MT targets based on thesingle mismatch between the reporter and the quencher which is near the5′ end. The 5′ portion of the SNP specific probe can hybridize moreefficiently to the MT template as compared to the WT template. When theSNP specific probe finds the WT target, the single mismatch to the WTtarget can prevent hybridization and probe cleavage, and thus nofluorescence can be detected.

In one embodiment, a method for detecting a SNP in a target nucleic acidin a sample is provided, the method including performing an amplifyingstep including contacting the sample with a first primer having a firstnucleic acid and a second primer having a second nucleic acid sequenceto produce an amplification product including a sense strand and ananti-sense strand if any target nucleic acid is present in the sample;performing a hybridizing step including providing the amplificationproduct with a double stranded probe including a first SNP specifichydrolysis probe having a third nucleic acid sequence complementary to afirst SNP containing region of the sense strand, the first SNP specifichydrolysis probe including a first interactive label and a secondinteractive label, a first 5′ end and a first 3′ end; and a second SNPspecific hydrolysis probe having a fourth nucleic acid sequencecomplementary to a SNP containing region of the anti-sense strand, thesecond SNP specific hydrolysis probe including a third interactive labeland a fourth interactive label, a second 5′ end and a second 3′ end, anddetecting the presence or absence of the amplification product, whereinthe presence of the amplification products is indicative of the presenceof the SNP in the target nucleic acid target, and wherein the absence ofthe amplification products is indicative of the absence of the SNP inthe target nucleic acid target. In some embodiments, the second SNPspecific hydrolysis probe can include a hairpin structure toward thesecond 3′ end, the hairpin structure including a region of non-naturallyoccurring nucleic acid sequence including one or more additionalnucleotides to produce the hairpin structure.

In another embodiment, a kit for detecting a SNP in a target nucleicacid in a sample is provided, the kit including a first primer having afirst nucleic acid and a second primer having a second nucleic acidsequence specific to produce an amplification product including a sensestrand and an anti-sense strand of a target nucleic acid; and a doublestranded probe including a first SNP specific hydrolysis probe having athird nucleic acid sequence complementary to a first SNP containingregion of the sense strand, the first SNP specific hydrolysis probeincluding a first interactive label and a second interactive label, afirst 5′ end and a first 3′ end; and a second SNP specific hydrolysisprobe having a fourth nucleic acid sequence complementary to a SNPcontaining region of the anti-sense strand, the second SNP specifichydrolysis probe including a third interactive label and a fourthinteractive label, a second 5′ end and a second 3′ end. In someembodiments, the second SNP specific hydrolysis probe can include ahairpin structure toward the second 3′ end, the hairpin structureincluding a region of non-naturally occurring (e.g., changed oradditional) nucleic acid sequence including one or more additionalnucleotides to produce the hairpin structure.

In one embodiment, a double stranded probe is provided including a firstSNP specific hydrolysis probe having a first nucleic acid sequencecomplementary to a first SNP containing region of the sense strand, thefirst SNP specific hydrolysis probe including a first interactive labeland a second interactive label, a first 5′ end and a first 3′ end; and asecond SNP specific hydrolysis probe having a second nucleic acidsequence complementary to a SNP containing region of the anti-sensestrand, the second SNP specific hydrolysis probe including a thirdinteractive label and a fourth interactive label, a second 5′ end and asecond 3′ end. The first and the third interactive labels may be a donorfluorescent moiety toward, near, or at the 5′ terminus of each probestrands, and the second and fourth interactive labels may be acorresponding acceptor fluorescent moiety, e.g., a quencher, forexample, within no more than 8 nucleotides of the donor fluorescentmoiety on each of the strands of the double stranded hydrolysis probe.In some embodiments, the second SNP specific hydrolysis probe caninclude a hairpin structure toward the second 3′ end, the hairpinstructure including a region of non-naturally occurring (e.g., changedor additional) nucleic acid sequence including one or more additionalnucleotides to produce the hairpin structure.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows real time PCR amplification curves for wild type and Mutant526N SNP detection using a double stranded hydrolysis probe with bothsense and antisense probe strands being SNP specific and the sensestrand having a hairpin structure toward the 3′ end.

FIG. 2 shows a 526N SNP specific double stranded hydrolysis probe withboth sense and antisense probe strands being SNP specific and the sensestrand having a hairpin structure toward the 3′ end.

FIG. 3 shows real time PCR amplification curves for wild type and Mutant531L SNP detection using a double stranded hydrolysis probe with bothsense and antisense probe strands being SNP specific.

FIG. 4 shows a 531L SNP specific double stranded hydrolysis probe withboth sense and anti-sense probe strands being SNP specific.

FIG. 5 shows real time PCR amplification curves for wild type and Mutant526Y SNP detection using a double stranded hydrolysis probe with bothsense and antisense probe strands being SNP specific.

FIG. 6 shows a 526Y SNP specific double stranded hydrolysis probe withboth sense and anti-sense probe strands being SNP specific.

DETAILED DESCRIPTION OF THE INVENTION

Methods, kits, and hydrolysis probes for detecting a single nucleotidepolymorphism (SNP) in a target nucleic acid in a sample are describedherein. The increased sensitivity of real-time PCR for detection of aSNP in a target nucleic acid compared to other methods, as well as theimproved features of real-time PCR including sample containment andreal-time detection of the amplified product, make feasible theimplementation of this technology for routine diagnosis and detection ofa SNP in a target nucleic acid in the clinical laboratory.

The methods may include performing at least one cycling step thatincludes amplifying one or more portions of a target nucleic acidmolecule, e.g., a gene target containing the SNP of interest to bedetected, in a sample using a primer including a nucleic acid andanother primer including another nucleic acid sequence to produce anamplification product including a sense strand and an anti-sense strand.As used herein, “primer”, “primers”, and “primer pairs” refer tooligonucleotide primer(s) that specifically anneal to the nucleic acidsequence target, and initiate synthesis therefrom under appropriateconditions. Each of the primers anneal to a region within or adjacent tothe respective target nucleic acid molecule such that at least a portionof each amplification product contains nucleic acid sequencecorresponding to respective target and SNP, if present. An amplificationproduct is produced provided that the target nucleic acid is present inthe sample, whether or not the SNP of interest is present in the targetnucleic acid molecule.

The method can also include a hybridizing step that includes providingthe amplification product with a double stranded probe including one SNPspecific hydrolysis probe including a nucleic acid sequencecomplementary to a SNP containing region of the sense strand of theamplification product, and another SNP specific hydrolysis probeincluding. another nucleic acid sequence complementary to a SNPcontaining region of the anti-sense strand of the amplification product.The double stranded SNP specific hydrolysis probes may be completelydouble stranded across the entire lengths of the sense and theanti-sense stands of the probes, or the double stranded probe may bepartially double stranded across a region of the lengths of the senseand the anti-sense strand of the probes. For example, the region wherethe sense and the anti-sense strands of the double stranded probe form adouble stranded region may be, for example, a region toward the 5′ end,or the 3′ end, or the central area of the sense and the anti-sensestrands of the double stranded probe. The double stranded probe shouldoverlap across the area where the SNP of interest is located. Each ofthe SNP specific hydrolysis probes of the double stranded probe caninclude a first and a second interactive label, a 5′ end and a 3′ end.In some embodiments, one hydrolysis probes of the double stranded probe,e.g., the anti-sense probes, can include a hairpin structure toward the3′ end. The hairpin structure can be designed to include a nucleic acidregion that is non-naturally occurring which may include one or morechanged nucleotides that are not part of the naturally occurringsequence, or may include one or more additional non-naturally occurringnucleotides, which are nucleotides added to the naturally occurringsequence, in order to produce the hairpin structure. In this way, anucleic acid sequence that does not normally form a hairpin structure atthe 3′end can be designed to form a hairpin by, e.g., altering thenucleic acid sequence, for example, changing one or more nucleotides inthe sequence toward the 3′ end, or by adding one or more nucleotides tothe nucleic acid sequence at the 3′end.

In order to detect whether or not the SNP of interest is present orabsent in the nucleic acid target in the sample, the amplificationproduct is detected by way of the detectable label being released fromboth of the first and second SNP specific hydrolysis probes. If theamplification product is detected by way of the double stranded SNPspecific hydrolysis probes, the presence of SNP is indicated. Ifalternatively, the amplification product is not detected by way of thedouble stranded SNP specific hydrolysis probes, the presence of SNP isnot indicated. Thus, the presence of the amplification products (senseand/or anti-sense) is indicative of the presence of the SNP in thetarget nucleic acid target, and the absence of the amplificationproducts is indicative of the absence of the SNP in the target nucleicacid target.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., target nucleicadd molecules for Human immunodeficiency virus (HIV) or Mycobacteriumtuberculosis (MTB), or Hepatitis C virus (HCV)). Amplifying a nucleicacid molecule typically includes denaturing the template nucleic acid,annealing primers to the template nucleic acid at a temperature that isbelow the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product.Amplification typically requires the presence of deoxyribonucleosidetriphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and anappropriate buffer and/or co-factors for optimal activity of thepolymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “primer” is used herein as known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′—OH group whereto further“nucleotides” may be attached by a template-dependent DNA polymeraseestablishing 3′ to 5′ phosphodiester linkage whereby deoxynucleosidetriphosphates are used and whereby pyrophosphate is released. Ingeneral, primers are designed based on known template sequences. Oneprimer primes the sense strand, and the other primes the complementary(anti-sense) strand of the target DNA or cDNA. PCR can be performed on auniform target DNA or RNA (i.e., targets with the same sequence) or onmixed target DNAs or RNAs, (i.e., targets with different interveningsequences flanked by conserved sequences). For mixed DNAs/RNAs (e.g.,containing sequence heterogeneity) even mismatched primers can functionin the PCR reaction if the sequences of the targets have enoughcomplementarity to the mismatched primers (i.e., tolerant primers).

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., aC5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides of thepresent disclosure are referred to herein or are otherwise known in theart. In certain embodiments, modified nucleotide substitutions modifymelting temperatures (Tm) of the oligonucleotides relative to themelting temperatures of corresponding unmodified oligonucleotides. Tofurther illustrate, certain modified nucleotide substitutions can reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like in some embodiments. Examples of these typesof nucleic acid modifications are described in, e.g., U.S. Pat. No.6,001,611, which is incorporated herein by reference.

As used herein, the term “non-naturally occurring nucleotide” in thecontext of the hairpin structure toward the 3′ end of the probe asdescribed herein, refers to a nucleotide that is not a naturallyoccurring nucleotide in the natural sequence, e.g., in the wild typesequence. Such non-naturally occurring nucleotide may be nucleotide thathas been changed, for example A to G, or may be an added non-naturallyoccurring nucleotide, for example a G may be inserted into the sequence.The inclusion of the non-naturally occurring nucleotides can be designedinto the natural sequence in order to produce the hairpin structure, inother words, the natural sequence can be engineered to force a hairpinstructure where a hairpin structure would not naturally occur. Thenatural sequence may be designed to include a hairpin structure towardthe 3′ end by changing or adding at least one non-naturally occurringnucleotide in the natural sequence, for example 1, 2, 3, 4, 5, 6, or 7non-naturally occurring nucleotides may be included in the naturalsequence in order to produce the hairpin structure.

Target Nucleic Adds and Oligonucleotides

The present description provides methods to detect SNP in a targetnucleic acid by amplifying, for example, a portion of the target nucleicacid sequences, which may be any target nucleic acid sequence known orsuspected to comprise one or more SNPs, for example target nucleic acidsequences from, e.g., HIV, HCV, or MTB that is rifampicin resistant.

For detection of SNP in the target nucleic acid sequence, primers andprobes to amplify the target nucleic acid sequences can be prepared.Also, functional variants can be evaluated for specificity and/orsensitivity by those of skill in the art using routine methods.Representative functional variants can include, e.g., one or moredeletions, insertions, and/or substitutions in the primers and/or probesdisclosed herein. For example, a substantially identical variant of theprimers or probes can be provided in which the variant has at least,e.g., 80%, 90%, or 95% sequence identity to one original primers andprobes, or a complement thereof.

A functionally active variant of any of primer and/or probe may beidentified which provides a similar or higher specificity andsensitivity in the presently described methods, kits, or hydrolysisprobes as compared to the respective original sequences.

As detailed above, a primer (and/or probe) may be chemically modified,i.e., a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify the target nucleic acid sequences can be designedusing, for example, a computer program such as OLIGO (Molecular BiologyInsights Inc., Cascade, Colo.). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis), similar melting temperatures forthe members of a pair of primers, and the length of each primer (i.e.,the primers need to be long enough to anneal with sequence-specificityand to initiate synthesis but not so long that fidelity is reducedduring oligonucleotide synthesis). Typically, oligonucleotide primersare 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

In addition to a set of primers, the present methods may use doublestranded probes in order to detect the presence or absence of SNP in atarget nucleic acid sequence. The term “probe” refers to syntheticallyor biologically produced nucleic acids (DNA or RNA), which by design orselection, contain specific nucleotide sequences that allow them tohybridize under defined predetermined stringencies specifically (i.e.,preferentially) to “target nucleic acids”. A “probe” can be referred toas a “detection probe” meaning that it detects the target nucleic acid.

In some embodiments, the described probes can be labeled with at leastone fluorescent label. In one embodiment probes can be labeled with adonor fluorescent moiety, e.g., a fluorescent dye, and a correspondingacceptor fluorescent moiety, e.g., a quencher.

Designing oligonucleotides to be used as TaqMan hydrolysis probes can beperformed in a manner similar to the design of primers. Embodiments ofthe present disclosure may use a double stranded probe for detection ofthe amplification product. Depending on the embodiment, the probe mayinclude at least one label and/or at least one quencher moiety. As withthe primers, the probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18,20, 21, 22, 23, 24, or 25) nucleotides in length.

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188 discloseconventional PCR techniques. U.S. Pat. Nos. 5,210,015; 5,487,972;5,804,375; 5,804,375; 6,214,979; and 7,141,377 disclose real-time PCRand TaqMan® techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the described embodiments includeoligonucleotides capable of acting as points of initiation of nucleicacid synthesis within the target nucleic acid sequences. A primer can bepurified from a restriction digest by conventional methods, or it can beproduced synthetically. The primer is preferably single-stranded formaximum efficiency in amplification, but the primer can bedouble-stranded. Double-stranded primers are first denatured, i.e.,treated to separate the strands. One method of denaturing doublestranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the target nucleicacid molecules. The temperature for annealing is usually from about 35°C. to about 65° C. (e.g., about 40° C. to about 60° C.; about 45° C. toabout 50° C.). Annealing times can be from about 10 sec to about 1 min(e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). Thereaction mixture is then adjusted to a temperature at which the activityof the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the template nucleic acid. The temperatureshould be sufficient to synthesize an extension product from each primerthat is annealed to a nucleic acid template, but should not be so highas to denature an extension product from its complementary template(e.g., the temperature for extension generally ranges from about 40° C.to about 80° C. (e.g., about 50° C. to about 70° C.; about 60° C.).Extension times can be from about 10 sec to about 5 min (e.g., about 30sec to about 4 min; about 1 min to about 3 min; about 1 min 30 sec toabout 2 min).

PCR assays can employ target nucleic acid such as RNA or DNA (cDNA). Thetemplate nucleic acid need not be purified; it may be a minor fractionof a complex mixture, such as target nucleic acid contained in humancells. Target nucleic acid molecules may be extracted from a biologicalsample by routine techniques such as those described in DiagnosticMolecular Microbiology: Principles and Applications (Persing et al.(eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 g denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acid molecules. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, a oligonucleotide probe can contain a donor fluorescentmoiety and a corresponding quencher, which may or not be fluorescent,and which dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between thetwo fluorescent moieties such that fluorescent emission from the donorfluorescent moiety is quenched. During an extension step of a polymerasechain reaction, a probe bound to an amplification product is cleaved bythe 5′ to 3′ nuclease activity of, e.g., a Taq polymerase such that thefluorescent emission of the donor fluorescent moiety is no longerquenched. Exemplary probes for this purpose are described in, e.g., U.S.Pat. Nos. 5,210,015; 5,994,056; and 6,171,785. Commonly useddonor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchersare DABCYL and TAMRA. Commonly used dark quenchers include BlackHoleQuenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Calif.), IowaBlack™, (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry™Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Mich.).

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a fiber optic light source, orother high intensity light source appropriately filtered for excitationin the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an absorbance spectrum that overlaps the emission spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present disclosure is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 Å to about 25 Å. The linker armmay be of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide which contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of a SNP in a Target Nucleic Acid

The present disclosure provides methods for detecting the presence orabsence of a SNP in a target nucleic acid in a biological. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of the target nucleic acidmolecule from a sample using a primer pair, and a fluorescent detectingstep utilizing double stranded SNP specific hydrolysis probes wherein insome embodiments one strand of the double stranded probes includes ahairpin structure toward the 3′ end. Multiple cycling steps may beperformed, preferably in a thermocycler. The described methods can beperformed using the primers and probes to detect the presence of the SNPin a target nucleic acid in the sample.

As described herein, amplification products can be detected usinglabeled hybridization hydrolysis probes that take advantage of FRETtechnology. One FRET format utilizes TaqMan® technology to detect thepresence or absence of an amplification product, and hence, the presenceor absence of the target nucleic acid. TaqMan® technology utilizes onesingle-stranded hybridization hydrolysis probe labeled with, e.g., onefluorescent dye and one quencher, which may or may not be fluorescent.When a first fluorescent moiety is excited with light of a suitablewavelength, the absorbed energy is transferred to a second fluorescentmoiety according to the principles of FRET. The second fluorescentmoiety is generally a quencher molecule. During the annealing step ofthe PCR reaction, the labeled hybridization probe binds to the targetDNA (i.e., the amplification product) and is degraded by the 5′ to 3′nuclease activity of, e.g., the Taq Polymerase during the subsequentelongation phase. As a result, the fluorescent moiety and the quenchermoiety become spatially separated from one another. As a consequence,upon excitation of the first fluorescent moiety in the absence of thequencher, the fluorescence emission from the first fluorescent moietycan be detected. By way of example, an ABI PRISM® 7700 SequenceDetection System (Applied Biosystems) uses TaqMan® technology, and issuitable for performing the methods described herein for detecting thepresence or absence of the target nucleic acid in the sample.

Generally, the presence of FRET indicates the presence of the SNP in atarget nucleic acid in the sample, and the absence of FRET indicates theabsence of the SNP in the sample. Inadequate specimen collection,transportation delays, inappropriate transportation conditions, or useof certain collection swabs (calcium alginate or aluminum shaft) are allconditions that can affect the success and/or accuracy of a test result,however. Using the methods disclosed herein, detection of FRET within,e.g., 45 cycling steps is indicative of the presence of an SNP in atarget nucleic acid in a sample.

Representative biological samples that can be used in practicing themethods of the present disclosure include, but are not limited to dermalswabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissueinfections. Collection and storage methods of biological samples areknown to those of skill in the art. Biological samples can be processed(e.g., by nucleic acid extraction methods and/or kits known in the art)to release target nucleic acid or in some cases, the biological samplecan be contacted directly with the PCR reaction components and theappropriate oligonucleotides.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996; 5,683,896; and 5,945,313 toreduce or eliminate contamination between one thermocycler run and thenext.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods of the present disclosure. In one embodiment, aLightCycler® instrument is used. The following patent applicationsdescribe real-time PCR as used in the LightCycler® technology: WO97/46707, WO 97/46714, and WO 97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

The present disclosure further provides for articles of manufacture orkits to detect a SNP in a target nucleic acid. An article of manufacturecan include primers and double stranded probes used to detect the SNP asdescribed herein, together with suitable packaging materials.Representative primers and probes for detection of the SNP are capableof hybridizing to the target nucleic acid molecules. In addition, thekits may also include suitably packaged reagents and materials neededfor DNA immobilization, hybridization, and detection, such solidsupports, buffers, enzymes, and DNA standards. Methods of designingprimers and probes are disclosed herein, and representative examples ofprimers and probes that amplify and hybridize to a SNP in a targetnucleic acid target nucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theSNP specific probes. Examples of suitable FRET donor fluorescentmoieties and corresponding acceptor fluorescent moieties are providedabove.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the primers and probes todetect a SNP in a target nucleic acid in a sample. Articles ofmanufacture may additionally include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes, co-factors,or agents to prevent contamination). Such reagents may be specific forone of the commercially available instruments described herein.

Embodiments of the disclosed subject matter will be further described inthe following examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example I MTB-RIF TaqMan® SNP Detection

Tuberculosis (TB) is a serious lung disorder commonly caused byMycobacterium tuberculosis (MTB) or other members of the MTB-complex.Drug-resistant strains of MTB are on the rise and a particularlydangerous form of drug-resistant tuberculosis is multidrug-resistanttuberculosis (MDR-TB). MDR-TB is defined as MTB that has developedresistance to at least two of the most commonly used anti-tuberculosisdrugs, rifampicin and isoniazid. Approximately 83-87% of the rifampicinresistance is caused by single nucleotide polymorphism within the 81base pair Rifampicin Resistance Determining Region (RRDR) of the rpoBgene encoding the β-subunit of RNA polymerase.

Mutant specific double stranded TaqMan® hydrolysis probes were designedwith a fluorophore at the 5′end and an internal quencher for each probestrands. Each of the sense and anti-sense strands of the probes weredesigned to be SNP specific, i.e., being perfectly matched with thesense and anti-sense mutant version of target containing the SNP ofinterest, and being similarly mismatched with the wild type version ofthe target. In addition, in some embodiments additional base/bases wereintroduced at the 3′end of the probe that would result in a hairpinstructure towards the 3′end. Probes were designed so that the mismatchbetween the WT and MT lies between the reporter and the quencher nearthe 5′end on each strand of the probes. When the TaqMan® probe isintact, the reporter and quencher stay close to each other, whichprevent the emission of any fluorescence.

The primer and TaqMan® probe anneal to the complementary DNA strandfollowing denaturation during PCR. After hybridization and during theextension phase of PCR, the 5′ to 3′ nuclease activity of the DNApolymerase cleaves the probe which separates reporter and quencher dyesand fluorescence is detected. In those embodiments which include thehairpin structure, the hairpin structure near the 3′end of the probedelays the hybridization of the 3′ half of the probe to the template andthus helps in the discrimination of the WT and the MT target based onthe single base pair difference or mismatch. The 5′ half of the MThairpin TaqMan® probe will hybridize more efficiently to the MT plasmidDNA template as compared to the WT template. When the MT specific probefinds the WT target, the single mismatch to the WT target will preventhybridization and probe cleavage and little or no fluorescence isdetected.

Materials and Methods

DNA Target—Wild-Type (WT) and Mutant (MT) Plasmids

MT and WT plasmid DNA: tested inputs ranging from 1e6 cp/PCR to 10cp/PCR shown in Table I showing a portion of the Rifampicin ResistanceDetermining Region (RRDR) of the rpoB gene in Mycobacteriumtuberculosis.

TABLE I Wildtype and Mutant Plasmid DNA SEQ ID NO SEQUENCE  7 MT PlasmidGCCAGCTGAGCCAATTCATGGTCCAGAAC with 526N SNPAACCCGCTGTCGGGGTTGACCCACAAGCG CCGACTGTCGGCGCTGGGGTCCGGCGG  8MT Plasmid for GCCAGCTGAGCCTATTCATGGACCAGAAC 531L SNPAACCCGCTGCAGGGGTTGACCCACAAGCG CCGACTGTTGGCGCTGGGGCCCGGCGG  9 MT PlasmidGCCAGCTGAGCCAATTCATGGACCAGAAC with 526Y SNPAACCCGCTGTCGGGGTTGACCCACAAGCG CCGACTGTCGGCGCTGGGGCCCGGCGG 10 WT PlasmidGCCAGCTGAGCCAATTCATGGACCAGAAC AACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGG

MTB specific oligonucleotides: One set of forward and reverse primersfor both wild-type and mutant plasmids

Mutant specific double stranded TaqMan® probes shown in Table II

TABLE II Sense and Antisense SNP Specific Probes SEQ  ID NO SEQUENCE 1Probe (526N SNP)- 5′-EALLAAQLAAGLGL Sense LGALP-3′ 2 Probe (526N SNP) 5′-ETTGTTGQGTCAAC with 3′ hairpin- CCCGAC GG G G P-3′ Antisense 3Probe (531L SNP)- 5′-EALUGUUQGGLGLU Sense GGP-3′ 4 Probe (531L SNP)-5′-ELLAALAQGTLGGL Antisense GLP-3′ 5 Probe (526Y SNP)- 5′-EALLUALQAAGLGLSense LGP-3′ 6 Probe (526Y SNP)- 5′-ELUUGUAQGGLLAA Antisense LLLLGAP-3′

Designations: E stands for Threo-HEX; P stands for Phosphate; Q standsfor BHQ-2 quencher, L stands for Propynyl dC; U stands for Propynyl dU;double underlined letters are the site of the SNPs; and boldedunderlined letters are bases that are changed or added to form hairpin.

Platforms: LightCycler® 480 System

Real time PCR amplifications were performed using a set of forward andreverse primers and double stranded SNP specific TaqMan® probes.Wild-type or Mutant plasmid DNA was tested at 1e6, 1e2, 1e3 and 1e1cp/PCR. The PCR reaction volume was 50 uL, and the master mix componentsand thermoprofile conditions are listed below. The amplifications wereperformed on the LC480 platform and the PCR growth curves were analyzedusing the ATF data analysis software. Results are shown in FIGS. 1through 6.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A method for detecting a single nucleotidepolymorphism (SNP) in a target nucleic acid in a sample, the methodcomprising: performing an amplifying step comprising contacting thesample with a first primer comprising a first nucleic acid and a secondprimer comprising a second nucleic acid sequence to produce anamplification product comprising a sense strand and an anti-sense strandif any target nucleic acid is present in the sample; performing ahybridizing step comprising providing the amplification product with adouble stranded probe comprising: a first SNP specific hydrolysis probecomprising a third nucleic acid sequence complementary to a first SNPcontaining region of the sense strand, the first SNP specific hydrolysisprobe comprising a first interactive label and a second interactivelabel, a first 5′ end and a first 3′ end; and a second SNP specifichydrolysis probe comprising a fourth nucleic acid sequence comprising aplurality of nucleotides complementary to a SNP containing region of theanti-sense strand, the second SNP specific hydrolysis probe comprising athird interactive label and a fourth interactive label, a second 5′ endand a second 3′ end; and detecting the presence or absence of theamplification product, wherein the presence of the amplificationproducts is indicative of the presence of the SNP in the target nucleicacid target, and wherein the absence of the amplification products isindicative of the absence of the SNP in the target nucleic acid target.2. The method of claim 1, wherein the second SNP specific hydrolysisprobe comprises a hairpin structure toward the second 3′ end, thehairpin structure comprising a region of non-naturally occurring nucleicacid sequence comprising one or more additional nucleotides to producethe hairpin structure.
 3. The method of claim 2, wherein the firstinteractive label comprises a first donor fluorescent moiety at thefirst 5′ terminus, and the second interactive label comprises a firstcorresponding acceptor moiety within no more than 8 nucleotides of thefirst donor fluorescent moiety on the first SNP specific hydrolysisprobe, and wherein the third interactive label comprises a second donorfluorescent moiety at the second 5′ terminus, and the fourth interactivelabel comprises a second corresponding acceptor moiety within no morethan 8 nucleotides of the second donor fluorescent moiety on the secondSNP specific hydrolysis probe.
 4. The method of claim 2, wherein thefirst acceptor moiety is a first quencher, and wherein the secondacceptor moiety is a second quencher.
 5. The method of claim 1, whereinthe amplification employs a polymerase enzyme having 5′ to 3′ nucleaseactivity.
 6. The method of claim 1, wherein the first and the secondnucleic acid sequences of the primers and/or the third and the fourthnucleic acid sequences of the hydrolysis probes comprise at least onemodified nucleotide.
 7. The method of claim 1, wherein the first and thesecond nucleic acid sequences of the primers and/or the third and thefourth nucleic acid sequences of the hydrolysis probes have 40 or fewernucleotides.
 8. A kit for detecting the presence or absence of a SNP ina target nucleic acid in a sample, comprising: a first primer comprisinga first nucleic acid and a second primer comprising a second nucleicacid sequence specific to produce an amplification product if the targetnucleic acid is present in the sample, the amplification productcomprising a sense strand and an anti-sense strand of a target nucleicacid; and a double stranded probe configured to detect the SNP if theSNP is present in the target nucleic acid, the double stranded probecomprising: a first SNP specific hydrolysis probe comprising a thirdnucleic acid sequence complementary to a first SNP containing region ofthe sense strand of the target nucleic acid, the first SNP specifichydrolysis probe comprising a first interactive label and a secondinteractive label, a first 5′ end and a first 3′ end; and a second SNPspecific hydrolysis probe comprising a fourth nucleic acid sequencecomprising a plurality of nucleotides complementary to a SNP containingregion of the anti-sense strand of the target nucleic acid, the secondSNP specific hydrolysis probe comprising a third interactive label and afourth interactive label, a second 5′ end and a second 3′ end.
 9. Thekit of claim 8, wherein the second SNP specific hydrolysis probecomprises a hairpin structure toward the second 3′ end, the hairpinstructure comprising a region of non-naturally occurring nucleic acidsequence comprising one or more additional nucleotides to produce thehairpin structure
 10. The kit of claim 9, wherein the first interactivelabel comprises a first donor fluorescent moiety at the first 5′terminus, and the second interactive label comprises a firstcorresponding acceptor moiety within no more than 8 nucleotides of thefirst donor fluorescent moiety on the first SNP specific hydrolysisprobe, and wherein the third interactive label comprises a second donorfluorescent moiety at the second 5′ terminus, and the fourth interactivelabel comprises a second corresponding acceptor moiety within no morethan 8 nucleotides of the second donor fluorescent moiety on the secondSNP specific hydrolysis probe.
 11. The kit of claim 9, wherein the firstacceptor moiety is a first quencher, and wherein the second acceptormoiety is a second quencher.
 12. The kit of claim 8, further comprisinga polymerase enzyme having 5′ to 3′ nuclease activity.
 13. The kit ofclaim 8, wherein the first and second nucleic acid sequences of theprimers and/or the third and fourth nucleic acid sequences of thehydrolysis probes comprise at least one modified nucleotide.
 14. The kitof claim 8, wherein the first and second nucleic acid sequences of theprimers and/or the third and fourth nucleic acid sequences of thehydrolysis probes have 40 or fewer nucleotides.
 15. A double strandedprobe configured to detect a SNP if the SNP is present in a targetnucleic acid, the double stranded probe comprising: a first SNP specifichydrolysis probe comprising a first nucleic acid sequence complementaryto a first SNP containing region of a sense strand of the target nucleicacid, the first SNP specific hydrolysis probe comprising a firstinteractive label and a second interactive label, a first 5′ end and afirst 3′ end; and a second SNP specific hydrolysis probe comprising asecond nucleic acid sequence comprising a plurality of nucleotidescomplementary to a SNP containing region of an anti-sense strand of thetarget nucleic acid, the second SNP specific hydrolysis probe comprisinga third interactive label and a fourth interactive label, a second 5′end and a second 3′ end.
 16. The double stranded probe of claim 15,wherein the second SNP specific hydrolysis probe comprises a hairpinstructure toward the second 3′ end, the hairpin structure comprising aregion of non-naturally occurring nucleic acid sequence comprising oneor more additional nucleotides to produce the hairpin structure
 17. Thedouble stranded probe of claim 16, wherein the first interactive labelcomprises a first donor fluorescent moiety at the first 5′ terminus, andthe second interactive label comprises a first corresponding acceptorfluorescent moiety within no more than 8 nucleotides of the first donorfluorescent moiety on the first SNP specific hydrolysis probe, andwherein the third interactive label comprises a second donor fluorescentmoiety at the second 5′ terminus, and the fourth interactive labelcomprises a second corresponding acceptor fluorescent moiety within nomore than 8 nucleotides of the second donor fluorescent moiety on thesecond SNP specific hydrolysis probe.
 18. The double stranded probe ofclaim 16, wherein the first acceptor fluorescent moiety is a firstquencher, and wherein the second acceptor fluorescent moiety is a secondquencher.
 19. The double stranded probe of claim 15, wherein first andsecond nucleic acid sequences of the hydrolysis probes comprise at leastone modified nucleotide.
 20. The double stranded probe of claim 15,wherein the first and second nucleic acid sequences of the hydrolysisprobes have 40 or fewer nucleotides.